Mutation of three critical amino acids of the N-terminal domain of IGF-binding protein-3 essential for high affinity IGF binding.

The N-terminal domain is conserved in all members of the IGF-binding protein superfamily. Most recently, studies have demonstrated the importance of an IGF-binding protein N-terminal hydrophobic pocket for IGF binding. To examine more critically the amino acids important for IGF binding within the full-length IGF-binding protein-3 protein while minimizing changes in the tertiary structure, we targeted residues I56, L80, and L81 within the proposed hydrophobic pocket for mutation. With a single change at these sites to the nonconserved glycine there was a notable decrease in binding. A greater reduction was seen when both L80 and L81 were substituted with glycine, and complete loss of affinity for IGF-I and IGF-II occurred when all three targeted amino acids were changed to glycine. Furthermore, the ability of the IGF-binding protein-3 mutants to inhibit IGF-I-stimulated phosphorylation of its receptor was a reflection of their affinity for IGF, with the lowest affinity mutants having the least inhibitory effect. These studies, thus, support the hypothesis that an N-terminal hydrophobic pocket is the primary site of high affinity binding of IGF to IGF-binding protein-3. The mutants provide a tool for future studies directed at IGF-dependent and IGF-independent actions of IGF-binding protein-3.

A 30-kDa fragment of insulin-like growth factor (IGF) binding protein-3 in human pregnancy serum with strongly reduced IGF-I binding.

Proteolytic cleavage of insulin-like growth factor (IGF) binding protein (IGFBP)-3 during pregnancy is likely to have both IGF-dependent and -independent effects on maternal, placental and fetal growth and metabolism. A 30-kDa proteolytic IGFBP-3 fragment was isolated from third trimester pregnancy human serum and identified by N- and C-terminal amino acid sequence analysis and mass spectrometry to correspond to residues 1-212 of the parent protein. This fragment is the dominating IGFBP-3 immunoreactive species in pregnancy serum. The 30-kDa fragment was also detected in serum of non-pregnant women where it coexists with intact IGFBP-3. Using biosensor technology, (1-212)IGFBP-3 was found to have 11-fold lower affinity for IGF-I compared to intact IGFBP-3, while a 4-fold decrease in affinity was found for IGF-II. Tests with des(1-3)IGF-I suggest fast binding of IGF-I to the N-terminal region of IGFBP-3 and similar affinity to a slow binding site in the C-terminal region.

Human skeletal muscle fibre type variations correlate with PPAR alpha, PPAR delta and PGC-1 alpha mRNA.

AIMS: Studies from genetically modified animals have been instrumental in highlighting genes and their products involved in the regulation of muscle fibre type and oxidative phenotypes; however, evidence in humans is limited. Our aim was therefore to investigate expression of those genes implicated in the regulation of oxidative fibre phenotypes in humans. METHODS: Using quantitative polymerase chain reaction we determined mRNA expression of selected genes in skeletal muscle from three different groups, displaying physiological and pathological variations in muscle fibre type, activity and skeletal muscle metabolism respectively: (i) elite athletes (cyclists), with an increased proportion of type I slow twitch, oxidative fibres, (ii) normally active subjects with an average fibre type distribution, and (iii) spinal cord-injured subjects with a low proportion of type I fibres. RESULTS: Skeletal muscle mRNA expression of calcineurin Aalpha and Abeta, peroxisome proliferator-activated receptor (PPAR)-alpha and -delta, and PPAR gamma coactivator (PGC)-1alpha and -1beta was determined. Calcineurin Aalpha and calcineurin Abeta mRNA expression was similar between groups. In contrast, mRNA expression of PPARalpha, PPARdelta, PGC-1alpha and -1beta was increased in athletes, when compared with normally active subjects. Furthermore, mRNA expression of PPARalpha, PPARdelta, PGC-1alpha and -1beta was reduced in spinal cord-injured subjects. Additionally, PPARalpha, PPARdelta and PGC-1alpha correlated with oxidative fibre content. CONCLUSION: Skeletal muscle mRNA expression of PPARalpha, PPARdelta, PGC-1alpha and -1beta reflects differences in type I muscle fibres associated with pathologically and physiologically induced skeletal muscle fibre type differences.

Free insulin-like growth factor I: are we hunting a ghost?

During the last decade, there has been an increasing number of publications reporting concentrations of free dissociable insulin-like growth factor I (IGF-I) in serum or plasma. The goal for attempting to measure free IGF-I in a serum sample in vitro has been to obtain information about the bioactivity of IGF-I in target tissues, and thus relate a measurable parameter to biological responses such as longitudinal growth or glucose disappearance rate. In this review, the serum free IGF-I approach is placed into a physiological perspective. In addition, methodological aspects are discussed and suggestions for the validation of free IGF-I assays are presented.